Direct Assembly and Metal-Ions Binding Properties of Oxytocin

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Direct Assembly and Metal-Ions Binding Properties of Oxytocin Monolayer on Gold Surfaces Evgeniy Mervinetsky, Israel Alshanski, Jörg Buchwald, Arezoo Dianat, Ivor Lon#ari#, Predrag Lazi#, Željko Crljen, Rafael Gutierrez, Gianaurelio Cuniberti, Mattan Hurevich, and Shlomo Yitzchaik Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b01830 • Publication Date (Web): 30 Jul 2019 Downloaded from pubs.acs.org on July 31, 2019

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Direct Assembly and Metal-Ions Binding Properties of Oxytocin Monolayer on Gold Surfaces Evgeniy Mervinetsky,1,2 Israel Alshanski,1,2 Jörg Buchwald,3 Arezoo Dianat,3 Ivor Lončarić,4 Predrag Lazić,4 Željko Crljen,*,4 Rafael Gutierrez,*,3 Gianaurelio Cuniberti,3,5,6 Mattan Hurevich,1,2 and Shlomo Yitzchaik*,1,2 Institute of Chemistry, and 2Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, E. Safra campus, Jerusalem 91904, Israel 3 Institute for Materials Science and Max Bergmann Center of Biomaterials, Hallwachsstraße 3, 01062 Dresden, Germany 4 Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia 5 Dresden Center for Computational Materials Science, TU Dresden, 01062 Dresden, Germany 6 Center for Advancing Electronics Dresden, TU Dresden, 01062 Dresden, Germany

1

E-mails: [email protected]; [email protected]; [email protected] KEYWORDS: Oxytocin, chemisorption, peptide, self-assembled monolayer, metal binding

ABSTRACT: Peptides are very common recognition entities which are usually attached to surfaces using multistep processes. These processes require modification of the native peptides and of the substrates. Using functional groups in native peptides for their assembly on surfaces without affecting their biological activity can facilitate the preparation of biosensors. Herein we present a simple single-step formation of native oxytocin monolayer on gold surface. These surfaces were characterizations by atomic force spectroscopy, spectroscopic ellipsometry and x-ray photoelectron spectroscopy. We took advantage of the native disulfide bridge of the oxytocin for anchoring the peptide to the Au surface, while preserving the metal ion binding properties. Self-assembled oxytocin monolayer was used by electrochemical impedance spectroscopy for metal ion sensing leading to sub-nanomolar sensitivities for zinc or copper ions.

Introduction Many peptides are natural binders of metal ions with distinct selectivity and specificity.1–4 Using peptides as biosensors for metal ions5–7 or as smart nano-materials for filtration8 and purification applications are an emerging and promising approaches.9 These applications require the attachment of the peptides to surfaces.10,11 Anchoring peptides to surfaces in a way that the inherent metal binding properties are maintained is a synthetic challenge, since in many cases this process requires chemical modifications of the peptide.12,13 Developing streamlined processes that will allow for the attachment of native peptides to surfaces without abolishing their metal binding properties is a valuable addition to the field. Using native molecular features in the peptide that are not involved in the biological function, e.g. metal ion binding, as anchoring moieties to the surface can guaranty that no modification on the peptide have to be made prior to the assembly. Oxytocin (OT) is an important hormone and neurotransmitter in the human body. OT is a nonapeptide which contains a six amino acid cycle and a short tail. The cycle contains a disulfide bond which is not involved in the metal ion binding. OT activity is regulated by binding to zinc and copper metal ion and has inherited affinity and selectivity to both ions.4,14,15 Zinc binding to OT enhances the affinity towards oxytocin receptor (OTR)15 hence increase the activity. Copper ions binding to OT takes place

via different binding groups and conformation and leads to reduced affinity toward OTR.1,16 These ions are important not only for OT binding regulation, but also for many biochemical and biophysical processes.17 Abnormal levels of these two ions can serve as an indicator for immunological and inflammatory disorders including autism, Alzheimer’s disease, multiple sclerosis, skin diseases, and cancer.18–23 OT based biosensing for zinc and copper can be very useful approach in the development of new line of diagnostics. The metal ion binding properties of OT depends on the peptide’s molecular features. It is crucial to preserve these molecular features in the preparation of OT based sensors. Recently, we reported a highly sensitive and selective OTderived electrochemical biosensor for Cu2+ and Zn2+ ions.13,24 The complexation of OT with these metal ions proceeds via different mechanisms: while OT-Cu complexation involves deprotonation of backbone and square-planar coordination, OT-Zn coordination proceeds with the amides’ carbonyl groups and forms a trigonal bipyramidal complex.14 In our previous reports, the oxide surface modification with an OT layer was conducted by a multi-step assembly process, which resulted in none native triazole-bridged OT. In order to allow for the attachment to surfaces and the preservation of the metal binding moieties an additional linker was added synthetically to the OT. We have searched for a simplified route for OT assembly on surfaces. Disulfides and thiols are known to coordinate very

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strongly to gold surfaces and organized into self-assembled monolayers (SAMs).25,26 Chemisorption of the disulfides on Au(111) surfaces results in dissociation of the disulfide bond and formation of two thiolate-gold bonds.27'28 OT is a peptide which is cyclized by a disulfide bond. We hypothesized that the native disulfide bond of OT can be used for anchoring the peptide to the Au surface without interfering with the metal ion binding abilities. Herein we present direct native OT monolayer chemisorption on a gold substrate. We investigate the peptide’s assembly and conformational changes upon metal ion binding applying electrochemistry, spectroscopic ellipsometry, X-ray photoelectron spectroscopy (XPS), Raman spectroscopy and Fourier transform infra-red spectroscopy (FTIR), and contact potential difference (CPD). We analyzed the metal binding potential of the new chemisorbed OT-Au layer to prove that it maintains the native chelation properties. Theoretical modeling was used to characterize the chemisorption and chelation mechanism.

Experimental methods OT-layer assembly. All solutions used in this work were prepared with Milli-Q Water (18.3 MΩ/cm, Millipore Milli-Q system (Bedford, MA)). Buffer solutions used were 50 mM ammonium acetate (pH=7.0) buffer (AAB) solution (Sigma-Aldrich). Gold surfaces were casted by 70 µL 250 μM OT (ProSpec-Tany Technogene Ltd., Israel) in AAB for 2 h, then rinsed with AAB and dried under dry N2 stream. Following peptide assembly the modified electrodes were exposed to Cu(NO3)2 (99.999%, trace metal basis, Sigma-Aldrich) and Zn(NO3)2 (99.999%, trace metal basis, Sigma-Aldrich) AAB solutions in different concentrations. Surface characterization. For surface characterization an Au layer (100 nm) was evaporated on top of Cr layer (10 nm), witch evaporated on the substrate of highly-doped ntype Si wafer (, R < 0.003 Ω/cm). OT layer was adsorbed on this substrate with the same adsorption protocol. Exposure to Cu2+/Zn2+ was done by drop casting of metal ions dissolved in AAB. XPS spectra were recorded using a monochromatic Al Kα X-ray source on a Kratos Axis-HS instrument. XPS analyses were applied to characterize the self-assembled peptide monolayers and the complexation of the peptide with Cu2+/Zn2+ ions. Raman Spectroscopy was collected with Renishaw inVia Reflex Spectrometer. The Au Surface Enhanced Raman Spectroscopy (SERS) substrates (SERStrate, Silmeco) were modified with OT layer and have been exposed to Cu2+/Zn2+ solution respectively. Polarization Modulation Infrared Reflection Absorption Spectroscopy (PMIRRAS) measurements were conducted at roomtemperature under positive nitrogen gas pressure on a reflection absorption cell (Harrick, Inc.) with PM-FTIR spectrometer (PMA-50 coupled to Vertex V70, Bruker). The signal collected from modified Au surfaces by 2048 scans with a resolution of 4 cm-1 using mercury cadmium telluride (MCT) detector. CPD measurements were performed with Kelvin probe S (DeltaPhi Besocke, Jülich, Germany), with a vibrating gold electrode (work function 5.1 eV) in a home-built Faraday cage under argon atmosphere. Variable angle spectroscopic ellipsometry (VASE) measurements were carried out with ellipsometer VB-400, Woollam Co. at the Brewster angle of 750. AFM

Page 2 of 10 (Bruker, Innova) was performed in tapping mode in order to monitor topography homogeneity of the layer as a result of OT adsorption and metal ion chelation. Electrochemical characterization. Electrochemical analyses were conducted with Bio-Logic SP-300 potentiostat (Bio-Logic Science Instruments, France), utilizing Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV) with EC-LAB software package. A three-electrode cell was used for the measurements: Ag/AgCl (in 3 M KCl) as reference electrode (RE), Pt as counter electrode (CE) and Au as working electrode (WE). Polycrystalline bulk gold electrodes with a 2 mm diameter were used for electrochemical measurements (CH instruments). These electrodes were manually-polished on micro-cloth pads (Buehler, Lake Bluff, IL) with de-agglomerated alumina suspension (Buehler) of decreasing particle size (1.0 and 0.05 µm) and washed with TDW. Then Au WE casted by OT by the described protocol. EIS measurements were performed at three stages: bare gold electrode, after adsorption of the peptide to the gold electrode and finally after exposure to metal ions. The frequencies range set from 100 kHz to 0.1 Hz with Ewe=0.21 V according Ag/AgCl reference electrode. All EIS scan were done in EIS solution of 5.0 mM K3[Fe(CN)6], 5.0 mM K4[Fe(CN)6] (RedOx species); and 0.1 M of KCl as supporting electrolyte in 50 mM AAB solution. The results fitted with the circuit RS[(RCT|W)||Q] where Q is constant phase element, that describes a non-ideal capacitor. For dose response, exposure of the Au-OT electrode proceeded by dipping of the Au-OT electrode in ion solution in AAB for 10 min, washing with the buffer (by rinsing the electrode with 1 ml AAB by Pasteur pipette), EIS measurement and further exposure for increasing concentrations of ions in AAB. Presented data based on statistics of 3 different electrodes. CV analysis was done on Au-OT electrode and Au-OT electrode after exposure to 10 µM Cu2+/AAB solution. The electrolyte solution was 0.1 M KCl in 50 mM AAB solution. The scan was done between -0.8V and +0.8V with various scan rates: 10 mV/s, 50 mV/s, 100 mV/s, 150 mV/s. Reductive desorption analysis26,29,30 performed for determination of surface coverage of OT on Au electrode. This characterization was done by CV in KOH [0.5 M] electrolyte with degassing by N2 for 5 min prior to the analysis. The CV recorded between -0.5 V and -1.4 V with scan rate of 100 mV/s. Computational methods Classical Molecular Dynamics. MD simulations were carried out using the force field developed by the Heinz group to describe the Au(111) surface.31 Therein, the interaction between Au atoms is described by a LennardJones potential. The force field parameters for oxytocin were computed with the aid of the Automated Topology Builder (ATB) to generate the necessary GROMOS force field parameters.32 For this purpose, we use fragmentation and the transferability of the GROMOS force field in order to obtain atomic charges from a Merz-Singh-Kollman (MK) scheme on the B3LYP/6-31G* level of DFT theory.33 The fragmentation was done such that every fragment was smaller than fifty atoms, and all fragments overlap each other. All MD simulations were performed using the GROMACS software. A periodic supercell with a size of


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74 nm³ was first equilibrated for 300 ps, followed by NoséHoover thermostating at 300 K within an NVT ensemble.34,35 Density-Functional Theory calculations. For the DFT calculations, a mixed Gaussian plane wave (GPW) method was used, as implemented in the CP2K software.36–38 For all DFT calculations, a mixed basis-set approach is used, where the Kohn-Sham orbitals are expanded into linear combinations of contracted Gaussian type orbitals (GTO), and complemented by a plane-wave basis set in order to compute the electronic charge density. The normconserving pseudo-potential GTH (Goedecker, Teter and Hutter) was used.39,40 In the case of Zn2+ binding to oxytocin, benchmarking showed that the LDA-PADE exchange-correlation functional was more appropriate for structural relaxation. For the work function calculations, however, the PBE functional was used. A DZVP (double zeta for valence electrons plus polarization functions) basis set complemented with a plane-wave basis set energy cutoff 350 Ry was further adopted. Nudge elastic band calculations (NEB) with climbing image method were performed with the ATK-DFT code using the PBE exchange-correlation functional with Grimme D2 dispersion corrections and SG15 pseudopotentials.41–45

Results and Discussion Direct chemisorption of the native neuropeptide OT was used on gold surface through disulfide bridge dissociation followed by S-Au bond formation (Figure 1). We have used various analytical methods to characterize the assembled monolayer and to evaluate the metal binding properties of the chemisorbed OT. O

NH2 NH2

O O

NH O

H NH O

S NH

S

HO O

O

O

NH HN

NH2

N

NH

HO

O

O

H

HN O

N H

NH NH

NH2 O

O O

NH2

HN O

NH

NH2 S

Au

NH2

O O O

O

HN

NH2

O NH

O

N S

O

Au

Figure 1. Chemisorption of OT via disulfide oxidative addition to Au surface forming Au-OT

Surface analysis. Anchoring of the OT to the gold surface through disulfide dissociation and S-Au bond forming was investigated first by surface enhanced Raman spectroscopy (SERS). SERS analysis was conducted on the Au-OT surface in order to characterize and identify the OT surface anchoring and ion-binding (Figure 2). A peak at 285 cm-1 was assigned to S-Au bond.46,47 This peak is significantly higher for Au-OT surfaces compared with bare Au SERS surface washed with AAB (Figure 2, black curve). The presence of the characteristic gold-sulfur bond energy by SERS and the absence of the disulfide bond related energy48 at 420-520 cm-1 (observed by ATR-FTIR of OT, see Figure S1) confirms chemisorption to gold by oxidative addition . SERS was also used to characterize peptide-ion interactions (Figure 2, Figure S2). An absorption in the 270-280 cm-1 spectral region was observed after exposure of the OT layer to copper or zinc ions. This new absorption is indicative of changes in sulfur-metal vibrations.49,50 A new absorption was observed at 389 cm-1 after exposure to copper. Since absorption at 350-400 cm-1 spectral region is usually

indicative of a deprotonated amide-Cu bonding,51,52 the Raman peak at 389 cm-1 (Figure 2, blue curve) can be related to N--Cu+2 bonding.

Figure 2. Surface enhanced Raman spectroscopy of bare Au (black), Au-OT (magenta), Au-OT after exposure to Cu2+ solution [10µM in AAB] (blue), Au-OT after exposure to Zn2+ solution [10µM in AAB] (red)

XPS analysis of samples with chemisorbed OT presents a significant peak at binding energies of 400 eV and 163.5 eV. These peaks related to N 1s (Figure S3) and S 2p (Figure S4) respectively. These signals are absent in scan of bare Au substrate. This presence of N and S peaks on the surface can only be attributed to the adsorption of OT to the Au substrate. Moreover, analysis of bare Au, represents only on C 1s-related peak at 284.2 eV (Figure S5). This peak may be attributed with some carbon impurities. However, analysis of Au-OT surfaces (Figure S6) clearly represents convolution of C 1s peaks at 284.8 eV, 286.06 eV and 288.13. These peaks indicated as C-C, C-N/C-O and O=C-N respectively53 and hence serve as an evidence for peptide presence of the surface. Ellipsometry analysis shows an organic layer thickness of 1.42 nm with good fit (MSE=3.8) for the OT layer on gold substrate. Ellipsometric analysis is in agreement with XPS thickness measurement of 1.41 nm for OT monolayer. These thickness analyses are compatible with the calculated cross-section of OT of ~1 nm, suggesting a stronger vdWmediated molecule-surface interaction compared to the experiments. Thickness measurements support OT monolayer formation with the longitudinal axis of the peptide lays in parallel to the surface. To measure surface roughness and verify the homogeneity of the surface, AFM analysis was performed for all modification steps (Figure S9). AFM shows that in all the assembly steps the surface topography is smooth and without defects or pin-holes. One can see surface smoothening following OT assembly54,55 and roughening following copper ion binding.5 The AFM analysis adds to the ellipsometry and XPS results proving the formation of OT monolayer. XPS was used to characterize the peptide layer upon the chelation with Cu2+/Zn2+ ions (see Figure S7, Figure S8). A peak at 1022.2 eV was observed only for the OT layer after exposure to Zn2+ ions. This peak is related to Zn2+ 2p 3/2 and is shifted by about -0.5 eV relative to the free-ion56 (Figure S7). A peak at 932 eV was observed after exposure of OT-Au to copper ions. (Figure S8) Such energy corresponds to the Cu2+ 2p 3/2 binding energy and is


Langmuir slightly shifted from the typical XPS Cu2+ peak (933.6 eV). This peak can be attributed to the chelation with the OT peptide by copper hence it is an indication that the peptide on the surface chelate the ion.57 XPS analysis of the nitrogen atom represents a single peak at 400.1 eV (Figure S3) which can be attributed to nitrogen in amide or amine bonds58. There is no spectral evidence for nitrogen at binding energy of about 407 eV characteristic for nitrogen in nitrate (N-O) residues. Cyclic Voltammetry analysis of OT layer with Cu2+. To confirm OT-Cu2+ chelation, a direct measurement of metal ions on the surface was performed by cyclic voltammetry (CV, see Figure 3). 0.006 0.004

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Au-OT-Cu2+ Au-OT

0.002 0.000 -0.002 -0.004 -0.006 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0

Voltage (V)

Figure 3. CV of Au-OT electrode (black) and Au-OT-Cu2+ electrode (red) prepared by exposure to 10 M Cu+2 solution. The CV was performed in KCl [0.1 M] in AAB [50 mM, pH=7] with scan rate of 100 mV/s.

By this analysis, we directly recognize an indicative Cu2+ oxidation peak at 0.26 V, which is significantly shifted from the oxidation peak of free Cu2+ ion (0.12 V). We can attribute the shift to OT-Cu complex formation.59 Moreover, CV with various scan rates (Figure S17) represents linear dependence of current to scan rate (Figure

Page 4 of 10 S18), which is characteristic for surface-bound redox centers. From the CV data, we were able to calculate the number of ions and surface concentration of the cooper ions in the layer according to Equation 1: 𝑄∗𝐶

𝑀𝑧 + = 𝐴 ∗ 𝑧

(1)

Here, M z+ is the amount of metal ions, z is a valence of the metal ions, Q is the charge transfer (calculated by the integral of the CV peak), C is the coulomb constant, and A is the area of the electrode. By calculating the amount of copper on the surface, we obtain a copper ions surface concentration of 0.75 ions/nm2 in the monolayer. Reductive desorption26,29,30 was done to measure the number of OT molecules. The characteristic peak at -1.1 V (Figure S15) indicates the reduction of S-thiol bond from Au surface. Since each OT molecule has two S-Au bonds, hence reduction of each molecule attributes with 2 electrons. The calculated footprint of OT is 2.76 nm2 (see SI), when the modelling of OT described 2.5 nm2. CV analysis yielded a ratio of Cu2+ to OT is 2:1 respectively. The CV analysis confirmed the presence of OT on the gold surface and the complexation between copper and the peptide. PM-IRRAS analysis of OT monolayer. Polarization modulation IR reflection-absorption spectroscopy measurements were performed to identify the changes in orientation of the specific functional groups. The intensity difference of the peaks represents dissimilar polarization of the band, namely the subtracting of spolarized beam, which is parallel to the surface of the sample from p-polarized beam, which is perpendicular to the sample surface.60 Hence, the alterations in PM-IRRAS spectra can be attributed to different molecular orientations,61 associated with the conformational changes in peptide layer. We used the PM-IRRAS to monitor conformational changes of the OT monolayer after ion binding (see, Figure 4).

Figure 4. PM-IRRAS: entire scan (A) and amide I region (B) of OT layer (black); OT after exposure to Cu2+ solution [10µM in AAB] (blue), and OT after exposure to Zn2+ solution [10µM in AAB] (red).


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We focused mostly on changes of signals in two regions, namely amide I and amide II. The amide I band (1600–1700 cm−1) is attributed to the C=O stretching vibration of the amide groups and to in-plane N-H bending. The amide II band (1480−1575 cm-1) derives mainly from in-plane N-H bending and from the C-N stretching vibration. Amide I spectral region is sensitive to the changes in peptide conformations, while the amide II band is less sensitive to these changes.62 Differences in OT spectra as function of exposure to metal ions are observed in the amide I (1620-1750 cm-1) and amide II (1530-1600 cm-1) regions. A significant increase in absorbance at 1745 cm-1 in the OT spectra was observed after exposure to Cu2+ and Zn2+. These indicate that significant conformational changes take place at the amide groups of the peptide monolayer. Interestingly, only after exposure of the monolayer to Zn2+ a sharp decrease in absorbance of the amide I region (at 1684-1726 cm-1) was observed (Figure 4B and Figure S10). Since this band is related to the orientation of the carbonyl groups, the observed change in orientation supports our hypothesis that OT binds zinc ions via carbonyl groups, in agreement with the suggested binding mechanism and peptide conformational changes. Additional changes were detected in 1480-1430 cm-1 (C-N groups63) and 1300-1500 cm-1 (amide fingerprint63) regions which further supports our assumption that these ion binding induces conformational changes (see also ATR spectra, Figure S1). Computational Modeling. In order to explore the representative conformational space of native OT as well as oxytocin on flat Au(111), we performed MD simulations at T=300 K. Moreover, we have also considered the influence of Cu2+ and Zn2+ binding to the OT conformation, both in the native state and on Au(111). The ability of free and surface adsorbed OT to bind Cu2+ and Zn2+ ions via deprotonated amides and via carbonyls, respectively, supports our experimental findings and is in good agreement with other studies.14 The results of the classical MD simulations are summarized in the Supplementary Information (Figure S11, Figure S12). In what follows, we exclusively focus on DFT-based calculations of the ionbinding energetics. Chemisorption of oxytocin on Au(111). State of the art knowledge of gold-sulfur interfaces identified a bridging Au atom between sulfur atoms as a key structural unit.64 Therefore, we also modeled oxytocin on Au(111) with one adatom as a minimal model of a more realistic surface. As shown in Figure 5, NEB calculations of the minimum energy path show that there is no significant barrier for dissociation of the disulfide bond and chemisorption of oxytocin over Au(111) with an adatom takes place (see also Movie S1). There is an energy gain of more than 4 eV per molecule upon chemisorption, and the Au adatom makes a bridge between the two S atoms64 with a S-Au bond of 2.3 Å and bonding angle S-Au-S of 136°. The absence of the dissociation barrier is a good explanation of the clear presence of the S-Au peak in Raman analysis presented in the experimental section.

Figure 5. Nudge elastic band calculation of the minimum energy path shows no barrier for disulfide bond dissociation on Au(111) with an adatom. The inset on the bottom left side shows the initial non-dissociated structure, while the top right inset shows the final structure with the broken disulfide bond.

Binding of Cu2+ to oxytocin. The binding cascade initiated by Cu2+ is addressed here, first for native OT. Cu2+ initially binds to the neutral N-terminal amino and afterwards to three additionally deprotonated amides before reaching the most stable square planar conformation. For the first reaction step in which Cu2+ binds to the amino group, we considered a small water cluster (three water molecules) near the Cu2+. Upon deprotonation of the amide groups and the formation of Cu-N bonds, the water molecules were subsequently removed from the simulation box.

Figure 6. Binding cascade of Cu2+ to native oxytocin.

The ion binding energy is computed as the total energy difference between the chelated system and the system in which ion + water is placed in the cell at a distance of about 20 Å from the OT molecule. The cascade reaction of Cu2+ is shown in Figure 6 for one- to four-fold coordination. We then addressed the binding of Cu2+ to OT attached to the gold surface. A summary of our results for free (native) OT and OT on the Au(111) surface is shown in Figure 7. The binding energy increases with increasing number of bonds between Cu2+ and the deprotonated amide groups for both cases, free OT and OT on the surface, although we found a slightly larger ion binding strength to OT on the surface. This can be attributed to the reduction of the conformational freedom due to the chemisorption process on the surface.


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Figure 7. Binding energies associated with the binding cascade of Cu2+ ions to free OT and to OT on the Au(111) surface.

Binding of Zn2+ to oxytocin. We have carried a similar analysis for Zn2+ binding, see Figure 8. We find that the ion binding coordination remained trigonal-bipyramidal, involving four carbonyls and the N-terminal amino. A binding energy of 0.09 eV was initially calculated using the PBE XC-functional. As this value seems to be unphysically low, we attribute the effect to be an artifact of the used GTH pseudopotential. Hence, we performed additional calculations using the LDA-PADE XC-functional taking only two valence electrons into account.39 Here, we obtained a rather high value of 8.47 eV. For the purpose of cross-checking, we further computed the corresponding binding energy for the Cu2+-chelation to OT using this functional and obtained a value of 6.75 eV, which is also higher than the value found with the PBE functional. These results are most likely related to an overestimation of the LDA exchange correlation effects, while the structural information nearly remains.

angles of about 100°, we concluded that Zn²⁺ chelates in a quasi-tetrahedral structure. A rather weak binding energy was obtained (binding energy of 1.68 eV), in clear contrast to the Cu²⁺ case (binding energy of 5.08 eV), see also Figure 8 and Table S1. We attribute this result to the more peripheral position of the Zn²⁺ ion as well as to the less stable tetrahedral chelation achieved on the surface (compared to the trigonal-bipyramidal chelation found for free OT with Zn²⁺in water). Work function changes upon ion binding. The work function (WF) change of the surface is directly proportional to the change in the surface electric dipole caused by the adsorption of molecules.65–67 Thereby, a strong correlation exists between the molecular dipole moments and the WF change induced by adsorbed molecules on metals and semiconductor surfaces.68–70 The WF is defined as the difference between electrostatic potentials at the vacuum level and at the Fermi energy of the surface. In Figure S13 and Figure S14 the corresponding WFs as a function of the z coordinate (perpendicular to the surface) are presented. The calculated WF of pure Au(111) of 5.28 eV is in very good agreement with other theoretical and experimental works.71 The presence of OT on the surface leads to a WF change dependent on its conformation. As we found quite different conformations in the MD simulation for the neutral OT and the deprotonated OT, we see that this transfers to the work functions as well. Therefore, we need to take these different WFs as reference points. In this case we find a decrease of the WF, which is of the same order of magnitude (See Table 1). Whereas Cu2+ binding leads to an increase of the WF by 0.76 eV, Zn2+ binding induces a WF reduction by 0.93 eV. The WF changes were experimentally determined by Kelvin Probe CPD. It was found, that after OT-Cu2+ chelation the WF increased by 49 meV, while OT-Zn2+ complexation resulted in decreasing of the WF by 54 meV (Figure 9). 80 60 40

CPD (meV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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20 0 -20 -40

Figure 8. Left panel: Free Oxytocin chelated in a trigonalbipyramidal structure to a Zn2+ via four carbonyls and the terminal amino. Right panel: Oxytocin on Au(111) with Zn2+ chelated in a trigonal bipyramid conformation.

For the surface attached OT, strong conformational changes seem to be restricted due to the interaction of the molecule with the surface. As a result, MD simulations showed that Zn²⁺ was less coordinated compared to the native OT case. After a DFT-based energy minimization, we found that Zn²⁺ dissolved from the terminal nitrogen and chelated with two other carbonyl oxygen atoms nearby. From the binding

-60

OT-Cu

OT-Zn

Figure 9. CPD measurements of Au-OT surface after exposure to copper ions (blue) and Au-OT after exposure to Zn2+ ions (red) These experimental observations are explained by the different chelation mechanisms of OT with Cu2+ and Zn2+. The formation of OT-Cu complex involves deprotonation of amide bonds, which cause a net negative charge (-1) of


Page 7 of 10 the complex. This results in an increase of the WF. However, the complex formation of OT-Zn does not go through deprotonation. Hence, the positive charge of the complex (+2) causes a decrease in WF. The theoretical calculations and experimental measurements displayed similar qualitative trends, albite some differences in the values. Our lower experimental values might be due to strong depolarization effects67 that are the result of intermolecular interaction in a monolayer diluting the net dipole and thus the change of the work function.72,73 Table 1. The calculated work function change upon ion binding. The indicated values are relative to Au(111) surface.

Au-OT

Au-OT-Zn2+

Au-OT-Cu2+

-0.48 eV

-1.41 eV

+0.28 eV

Dose response of OT to copper and zinc ions measured by EIS. To evaluate the interaction of OT monolayer with copper and zinc ions, OT-Au electrodes were introduced to various concentrations of the metal ions and the EIS response been measured (Figure S19, Figure S20). The measured RCT values were normalized to initial RCT value of the electrode before exposure to metal ions. Exposure time to the metal ion solution was relatively long (10 min) in order to nullify the influence of kinetics. From these results, a plot of normalized RCT values as a function of the ion concentration was made to represent the dose response of the sensing device (Figure 10).

1.8

Normalized Rct

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Zn Cu

1.6

1.4

1.2

1.0 10-14 10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

[M2+] (Molar)

Figure 10. EIS-derived dose response of OT sensor to Cu2+ ions (blue curve) and Zn2+ ions (red curve). RCT values after exposure to metal ion solutions normalized by the initial RCT. The frequencies range set from 100 kHz to 0.1 Hz with Ewe=0.21 V according Ag/AgCl reference electrode. All EIS scans were done in EIS solution of 5.0 mM K3[Fe(CN)6], 5.0 mM K4[Fe(CN)6] (RedOx species); and 0.1 M of KCl as supporting electrolyte in 50mM AAB solution. The OT sensor showed sensitivity to both metal ions in a wide range of concentrations. For Cu2+, the dynamic range observed for concentrations of 10-13 M to 10-9 M. For Zn2+ ions, the sensitivity detected from 10-13 M to 10-3 M. The increasing of RCT values resulted from conformational changes of peptide layer upon metal ion binding. Saturation

region of OT-Cu response from nM to mM region is achieved at 1:2 ratio between OT to copper ions, as suggested by CV analysis, while OT-Zn coordination through carbonyl groups didn’t exhibit saturation suggesting complexation that approaches 1:1 ratio. The label-free metal-ion sensing described above proved that using native OT attached to gold substrate can be useful for biosensing applications.

Conclusions In this study, we presented a new strategy for the assembly of native neuropeptide on gold surfaces. We developed a simple method for single-step fabrication of OT monolayers on gold substrates. We used an unmodified native peptide and utilized the disulfide group for direct anchoring to the surface and its other functional groups for metal ion binding. The conformational changes of the peptide layer, resulting from chelation of OT with metal ions were confirmed and characterized by electrochemical, spectral and theoretical calculations analysis. In this work, we showed that direct adsorption of native OT via disulfide bond to step edges regions of the gold surface allowed for the preservation of the peptide functionality. Furthermore, the absence of a dissociation barrier of the disulfide bond of OT on the gold surface with an adatom supports the experimental claim that OT is chemisorbed on Au via AuS bonds. Our study suggests that the assembly strategy preserves native metal ion chelation properties. This indicates that native neuropeptides immobilized on surface can be a very useful biomimetic tool.74 Our strategy could be applied for studying biorelevant interactions of this extremely important neuropeptide. The use of atomistic methodologies complementing the experimental studies have helped to shed light into the various possible coordination environments of Cu+2 and Zn+2 upon binding to OT on the Au substrate as well as to highlight the differences to the native (i.e., free) OT. The computed binding energies of Cu+2 and Zn+2 to OT on Au(111) suggest a stronger binding of Cu+2 to OT. This work shows that using native functional groups of peptides for direct assembly on gold can be highly useful for novel bioelectronic architectures and neuromorphic computing. The envisioned application of this model system can be extended for interaction of OT with OT-receptor probing and study other binding proteins in the presence and absence of different metal ions. In addition to flat surfaces this model can be used on nano-particles for bio-imaging applications.

Author Information Corresponding Authors: *E-mail: [email protected](S.Y.) *E-mail: [email protected] (Ž.C.) *E-mail: [email protected] (R.G.)

Author Contributions The manuscript was written through contributions of all


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authors. All authors have given approval to the final version of the manuscript. Funding Sources This investigation was carried out with financial support of the H2020-FETOPEN project Reservoir Computing with Real-time Data for future IT (RECORD-IT) under grant nr. 664786. IL, PL, and ŽC were also supported by the European Union through the European Regional Development Fund the Competitiveness and Cohesion Operational Programme (KK.01.1.1.06) and the H2020 CSA Twinning project No. 692194, RBI-T-WINNING.

Additional Information: Supplementary information: the following files are available free of charge. Supplementary figures and tables, AFM, SERS, ATR-FTIR, DFT calculations, additional electrochemical data, XPS analysis.

Notes

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The authors declare no competing financial interests.

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Acknowledgments

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SY is the Binjamin H. Birstein Chair in Chemistry. This research has also been partly supported by the German Research Foundation (DFG) within the Cluster of Excellence “Center for Advancing Electronics Dresden.”. TU Dresden gratefully acknowledged computing time by the Center for Information Services and High Performance Computing (ZIH). The authors would like to thank Dr. Vitaly Gutkin for XPS analysis; Shahar Dery and Dr. Elad Gross for FTIR analysis.

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Direct Assembly and Metal-Ions Binding Properties of Oxytocin

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